Improved proteome coverage and reproducibility in large cohort analyses using packed emitter columns

Proteomic analyses of large cohorts of human patient samples are used to understand the biological mechanisms of disease and to discover novel biomarkers for diagnosis and response to treatment. Deep proteome coverage and reproducibility of results is crucial for the success of these studies. We developed a novel packed emitter chromatography column, the IonOpticks Aurora Rapid 8x150 (8 cm length x 150 μm inner diameter, 1.7 μm C18), that improves the results from patient cohort analyses by ensuring high and consistent proteome coverage and reproducible quantitation. This study evaluates the efficiency of the column for high-throughput proteomic analysis of large patient plasma cohorts coupled with an Evosep One and a Thermo Fisher Orbitrap Astral mass spectrometer. As part of this study, patient plasma samples were analysed using a 100 samples per day method with approximately 700 samples run between quality control (QC) injections. To ensure consistent performance and reproducibility, the system underwent weekly QC injections of HeLa tryptic peptides using a 60 samples per day method. The system achieved robust protein identifications, with over 9,300 proteins and 100,000 unique peptides consistently identified per QC run across multiple columns. To assess the reproducibility of protein quantification across multiple QC runs and columns, protein intensities were used to calculate a coefficient of variation (CV) and Pearson correlation, revealing uniform performance with low CV values and high correlation between runs and between columns. This consistent performance, along with consistently narrow peak widths averaging 2.9 seconds FWHM, indicates good column performance and reproducibility, important for large patient cohort analyses. Furthermore, to ensure reliable data analysis, we evaluated retention time stability. This analysis demonstrated a high level of consistency, with a 2.52% median CV of retention times. Additionally, a four-month experiment on 16 selected peptides demonstrated stable retention times. Taken together, these results demonstrate that our novel packed emitter column combined with the Evosep One and Thermo Fisher Orbitrap Astral Mass Spectrometer enabled maximum protein and peptide identifications whilst also ensuring robust and reproducible protein quantification across a large patient plasma cohort analysis.